title: Binding of phospholipid transfer protein (PLTP) to apolipoproteins A-I and A-II: location of a PLTP binding domain in the amino terminal region of apoA-I.
abstract: The interaction of plasma phospholipid transfer protein (PLTP) with HDL has not been characterized in detail, although we have reported that the apoA-I/apoA-II molar ratio in the HDL particle influences PLTP-mediated HDL conversion, but not phospholipid transfer. The aim of this study was to examine whether PLTP binds apoA-I or apoA-II, and if this occurs, then determine the PLTP-binding domain of the apoA-I molecule. To study the PLTP/apolipoprotein interaction we used a solid phase ligand binding assay, the ELISA technique, and apoA-I and apoA-II affinity chromatography. PLTP bound to both apoA-I and apoA-II affinity columns, a finding subsequently utilized in the purification of PLTP. PLTP also bound to both apoA-I and apoA-II on ELISA plates in a concentration-dependent manner, and the binding could be displaced by preincubating the PLTP sample with purified apolipoproteins. To determine which portion of apoA-I is recognized by PLTP, we coated ELISA plates with either recombinant full-length apoA-I or three shortened apoA-I forms sequentially truncated from the C-terminus. To characterize the PLTP binding ability of the C-terminal region of apoA-I, we used both C-terminal CNBr-fragment and a synthetic C-terminal peptide of apoA-I. To further confirm the identity of the binding region, we probed the interaction with a polyclonal and several monoclonal anti-apoA-I antibodies. The antibodies that inhibited the interaction between PLTP and apoA-I were directed towards apoA-I epitopes localized between amino acids 27-141. The polyclonal antibody, R33, and the monoclonal antibody A-I-1 (epitope between amino acids 27-48) were most effective and reduced PLTP binding by 70%. These results show that PLTP binds to both apoA-I and apoA-II, and that the PLTP binding domain of apoA-I resides in the amino terminal region.
authors.affiliation: National Public Health Institute, Department of Biochemistry, Helsinki, Finland.
authors.forename: P J
authors.initials: PJ
authors.lastname: Pussinen
authors.forename: M
authors.initials: M
authors.lastname: Jauhiainen
authors.forename: J
authors.initials: J
authors.lastname: Metso
authors.forename: L E
authors.initials: LE
authors.lastname: Pyle
authors.forename: Y L
authors.initials: YL
authors.lastname: Marcel
authors.forename: N H
authors.initials: NH
authors.lastname: Fidge
authors.forename: C
authors.initials: C
authors.lastname: Ehnholm
mesh_terms: D000906:Antibodies; D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism*; D016633:Apolipoprotein A-II / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D049294:Phospholipid Transfer Proteins; D011994:Recombinant Proteins
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D002352:Carrier Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D049294:Phospholipid Transfer Proteins; D011994:Recombinant Proteins; D003488:Cyanogen Bromide